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Essential Oils of Cupressus funebris, Juniperus communis, and J. chinensis

(Cupressaceae) as Repellents Against Ticks (Acari: Ixodidae) and Mosquitoes
(Diptera: Culicidae) and as T...

Article  in  Journal of Vector Ecology · December 2011

DOI: 10.1111/j.1948-7134.2011.00166.x · Source: PubMed

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258 Journal of Vector Ecology December 2011

Essential oils of Cupressus funebris, Juniperus communis, and J. chinensis

(Cupressaceae) as repellents against ticks (Acari: Ixodidae) and mosquitoes
(Diptera: Culicidae) and as toxicants against mosquitoes
John F. Carroll1*, Nurhayat Tabanca2, Matthew Kramer3, Natasha M. Elejalde4, David E. Wedge2,
Ulrich R. Bernier4, Monique Coy4, James J. Becnel4, Betul Demirci5, Kemal Husnu Can Başer5,
Jian Zhang6, and Sui Zhang7
1
USDA, ARS, Invasive Insect Biocontrol and Behavior Laboratory, Beltsville Agricultural Research Center,
Beltsville, MD 20705, U.S.A. john.carroll@ars.usda.gov
2
USDA, ARS, Natural Products Utilization Research Unit, University of Mississippi, University, MS 38677, U.S.A.
3
USDA, ARS, Biometrical Consulting Service, Beltsville Agricultural Research Center, Beltsville, MD 20705, U.S.A.
4
USDA, ARS, Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL 32608, U.S.A.
5
Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470, Eskisehir, Turkey
6
School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai 200235, China
7
Plant Protection Institute, Shanghai Academy of Agricultural Sciences, Shanghai, 201106 China

Received 28 September 2010; Accepted 22 December 2010

ABSTRACT: Juniperus communis leaf oil, J. chinensis wood oil, and Cupressus funebris wood oil (Cupressaceae) from
China were analyzed by gas chromatography and gas chromatography-mass spectrometry. We identified 104 compounds,
representing 66.8-95.5% of the oils. The major components were: α-pinene (27.0%), α-terpinene (14.0%), and linalool
(10.9%) for J. communis; cuparene (11.3%) and δ-cadinene (7.8%) for J. chinensis; and α-cedrene (16.9%), cedrol (7.6%),
and β-cedrene (5.7%) for C. funebris. The essential oils of C. funebris, J. chinensis, and J. communis were evaluated for
repellency against adult yellow fever mosquitoes, Aedes aegypti (L.), host-seeking nymphs of the lone star tick, Amblyomma
americanum (L.), and the blacklegged tick, Ixodes scapularis Say, and for toxicity against Ae. aegypti larvae and adults, all
in laboratory bioassays. All the oils were repellent to both species of ticks. The EC95 values of C. funebris, J. communis, and
J. chinensis against A. americanum were 0.426, 0.508, and 0.917 mg oil/cm2 filter paper, respectively, compared to 0.683 mg
deet/cm2 filter paper. All I. scapularis nymphs were repelled by 0.103 mg oil/cm2 filter paper of C. funebris oil. At 4 h after
application, 0.827 mg oil/cm2 filter paper, C. funebris and J. chinensis oils repelled ≥80% of A. americanum nymphs. The oils
of C. funebris and J. chinensis did not prevent female Ae. aegypti from biting at the highest dosage tested (1.500 mg/cm2).
However, the oil of J. communis had a Minimum Effective Dosage (estimate of ED99) for repellency of 0.029 ± 0.018 mg/cm2;
this oil was nearly as potent as deet. The oil of J. chinensis showed a mild ability to kill Ae. aegypti larvae, at 80 and 100% at
125 and 250 ppm, respectively. Journal of Vector Ecology 36 (2): 258-268. 2011.

Keyword Index: Lone star tick, Amblyomma americanum, blacklegged tick, Ixodes scapularis, yellow fever mosquito, Aedes
aegypti, repellency, toxicant.

INTRODUCTION high rates of human morbidity and mortality.

Repellents applied to skin or clothing are recommended
Mosquito and tick-borne illnesses exact a considerable as a means of personal protection against biting arthropods
toll in human misery and financial expenditure. Problems (e.g., CDC 2002). Toxicants can reduce the densities of
with ticks and tick-borne diseases have continued to grow mosquito and tick populations and the risk of disease
during the past three decades (Parola and Raoult 2001). transmission. The synthetic repellent deet (N,N-diethyl-3-
Lyme disease (caused by the spirochete Borrelia burgdorferi) methyl benzamide) has been used widely since the 1950s
and its principal vector, Ixodes scapularis Say (Spielman et to defend against mosquitoes and ticks. Newer synthetics,
al. 1985), have deservedly received significant attention such as picaridin and IR3535, now provide more options for
in the United States. The lone star tick, Amblyomma users (Debboun et al. 2007).
americanum (L.), has acquired notoriety not only as a Permethrin, a synthetic pyrethroid, is approved to
nuisance biter, but also as a vector of Ehrlichia chaffeensis, be used as a repellent, but only when applied on clothing
which causes human monocytic ehrlichiosis (Childs and (Schreck et al. 1982). As toxicants, pyrethroids are used
Paddock 2003, Armstrong et al. 2001). Mosquitoes are to control a wide range of arthropods that are agricultural
vectors for many pathogens that cause serious diseases, and public health pests (Hoel et al. 2010). Repeated use
such as malaria, dengue fever, yellow fever, Rift Valley fever, of pyrethroids, or insecticides of any class, can lead to
and Chikungunya, which can attain epidemic levels with insecticide resistance. Consequently, there is an urgent
Vol. 36, no. 2 Journal of Vector Ecology 259

need to develop alternatives to chemical control of a wide Mosquitoes

variety of arthropod vectors of human diseases (Pridgeon Pupae of Ae. aegypti from the Gainesville (CMAVE)
et al. 2008, 2009). Many naturally occurring repellents and colony were maintained in the laboratory at 28 ±1° C and
insecticides have potential for development into useful 30-60% RH, and the resulting adults aged five to nine days
products because they combine efficacy, biodegradability, were used for repellent testing. The A. aegypti mosquitoes
and limited risk to mammals and the environment (Dayan (also from the CMAVE colony) used in the insecticide
et al. 2009, Hoel et al. 2010, Carter 1989, Bissinger et al. bioassays were held at 22-30° C, 80% RH, and a photoperiod
2009, Bissinger and Roe 2010). Plant essential oils and seed of 14:10 (L:D) including 2 h of simulated dusk and dawn.
pressed oils comprise a significant portion of the market The mosquitoes were maintained on 10% sucrose ad libitum
share for natural product-based insecticides, and some have and provided twice weekly with bovine blood in 1% heparin
served as the basis of commercial repellent formulations placed in pig intestine and warmed to 37° C.
(Dayan et al. 2009, Nerio et al. 2010).
A great diversity of medicinal and aromatic plants Essential oils, repellent, and insecticide
occurs in China, where these natural products exert a Juniperus communis oil was purchased from Shanghai
strong impact on peoples’ lives and culture (Li et al. 2006). Qika Corporation, Shanghai, China, and C. funebris and J.
Prominent among the useful taxa is the cypress family, chinensis oils were purchased from Peking University Zoteq
Cupressaceae. Commercial cedarwood oils have been Co., Ltd., Beijing, China. Deet was purchased from Aldrich,
obtained from three genera of Cupressaceae: Juniperus Sigma-Aldrich, Inc., St. Louis, MO, and permethrin from
(Texas, Virginia, and Africa), Cupressus (China), and Chem Service, West Chester, PA.
Cedrus (Morocco, India) (Adams and Li 2008). Cupressus
funebris Endl. is generally regarded as the botanical source GC/FID and GC/MS analysis
of Chinese cedarwood oil, a well known perfume material The oils were analyzed by capillary GC-FID and GC/MS
(Adams and Li 2008), and its derivatives (Duquesnoy et al. using an Agilent 5975 GC-MSD system (SEM Ltd., Istanbul,
2006). Turkey). A Hewlett Packard-Innowax FSC column (60 m
The studies of Panella et al. (1997, 2005), Dietrich et x 0.25 mm, 0.25 mm film thickness) was used with helium
al. (2006), and Dolan et al. (2007, 2009) indicate that the as the carrier gas (0.8 ml/min). The GC oven temperature
Cupressaceae may be a rich source of anti-tick compounds was held at 60° C for 10 min and ramped to 220° C at a rate
and that further investigation of cypress species for repellent of 4° C/min, then held constant at 220° C for 10 min with
and insecticidal chemicals is warranted. We investigated a final programmed ramp to 240° C at a rate of 1° C/min,
Chinese weeping cedar, C. funebris, Chinese juniper, and held a second time at 240° C for 20 min. The split ratio
Juniperus chinensis L., and common juniper, J. communis L., was adjusted to 40:1. The injector temperature was at 250°
essential oils from China. One purpose of this study was to C. The mass spectrometer was operated with an electron
characterize the chemical composition of the essential oils energy of 70 eV. The mass range was m/z 35 to 425 at a scan
of C. funebris, J. chinensis, and J. communis by analysis using rate of 3.46 scans/s. The GC analysis was carried out using
gas chromatography (GC) and GC-mass spectrometry an Agilent 6890N GC system equipped with a FID detector
(GC-MS). The second purpose was to evaluate these oils as operated at a temperature of 300° C. To obtain the same
repellents against the ticks A. americanum and I. scapularis elution order of peaks detected by GC/MS, simultaneous
and as repellents and adult and larval toxicants against the injection on the GC was performed using the same column
yellow fever mosquito, Aedes aegypti (L.). and appropriate chromatographic conditions as those
described for the GC/MS system.
MATERIALS AND METHODS Identification of the essential oil components was
carried out by comparing their relative retention times with
Ticks those of authentic samples or by comparing their relative
Larvae of I. scapularis from a colony at Oklahoma State retention index (RRI) to a series of n-alkanes. Computer
University were fed as larvae on rats at the USDA, ARS, matching identified compounds used as references (Wiley
Beltsville Agricultural Research Center, Beltsville, MD in and MassFinder 3.1) (Koenig et al. 2004, McLafferty and
accordance with USDA, ARS, Beltsville Area Animal Care Stauffer 1989). An in-house “Başer Library of Essential
and Use Committee Protocol #08-013. The fed larvae and Oil Constituents” composed of genuine compounds and
resultant nymphs were held at 23-24° C, ~97% RH and a components of known oils, and MS literature data (Joulain
photoperiod of 16:8 h (L:D). The A. americanum nymphs and Koenig 1998, ESO 2000, Jennings and Shibamoto
were from a colony at the USDA, ARS, Knipling-Bushland 1980) were also used for the identification. The relative
U. S. Livestock Insects Research Laboratory, Kerrville, TX concentrations of the separated compounds based on
and a colony at Oklahoma State University, Stillwater, OK percentage were computed from chromatograms obtained
(used in duration bioassay) and held at 23-24° C, ~97% RH with the GC/FID system.
and a photoperiod of 16:8 h (L:D). The I. scapularis and A.
americanum nymphs were tested three to five, and three to Vertical filter paper bioassay
six months after molting, respectively. Host-seeking ticks of many species tend to climb
vertical surfaces, particularly in the presence of host-
260 Journal of Vector Ecology December 2011

produced stimuli. This behavior was used to expose A. Mosquito repellent assay
americanum nymphs to repellent treatments in an in vitro Female mosquitoes were selected from the stock
bioassay described in detail by Carroll et al. (2004). Briefly, cages by a hand-draw box (Posey and Schreck 1981).
a 4 X 7-cm rectangle of Whatman No. 4 filter paper was Approximately 500 (± 10%) mosquitoes, primarily females,
marked with a pencil into two 1 X 4-cm zones at the far were transferred into a test cage (capacity 59,000 cm3,
ends of the paper strip and a central 4 X 5-cm zone. Using dimensions 45 cm x 37.5 cm x 35 cm) and held therein for
a pipettor, 165 µl of test solution was evenly applied to 25 (± 2.5) min before initiating repellency assays (Barnard
both sides of the central 4 X 5 cm of the filter paper. After et al. 2007).
drying for 10-15 min, the paper strip was suspended from Extracts were weighed and placed in a 2-dram vial to
a bulldog clip hung from a slender horizontal dowel held which 2 ml acetone was added. The initial weight of extract
by an Aptex No. 10 double clip work holder (Aptex, Bethel, was measured so that when one half (1 ml of solution)
CT). A Petri dish (9 cm diameter) glued in the center of was removed and a 50 cm2 muslin cloth was added to the
a 15 cm Petri dish created a moat when water was added vial, the remaining 1 ml solution would produce an initial
between their walls (1.5 cm high). The moated Petri dishes concentration on cloth of 1.500 mg/cm2. Serial dilutions
were placed directly beneath the suspended filter paper. were then made analogously such that the concentrations
When A. americanum nymphs climbed to the rim of a on cloth for the remaining 1 ml solution were: 0.750, 0.375,
storage vial opened in the center of the moated Petri dishes 0.187, 0.094, 0.047, 0.023, 0.011, and 0.005 mg/cm2. Vials
(5.5 and 9 cm diameters), the paper strip was removed from were sealed and stored at -4° C in a freezer until testing
the dowel and held so that ten ticks crawled onto the lower (normally <48 h). Each test involved removal of cloth from
untreated zone. Because nymphs of I. scapularis tended to the vial and stapling it onto two sections of card stock (5 cm
be slower and more apt to drop from untreated filter paper, x 2.5 cm). Pieces of masking tape (2.5-5.0 cm long) secured
they were screened for readiness to crawl while the test the cloth onto the card stock. The card and cloth assembly
solution dried on the filter paper (Schreck et al. 1995). As was then placed on a drying rack for 3-5 min before testing.
the treated filter paper dried, I. scapularis were transferred A single test consisted of covering the hand of a
with forceps to an untreated vertical filter paper. Ten ticks volunteer with a soft-embossed long cuff poly glove
that climbed >5 mm were selected for testing and placed in (Atlantis Products, Mankato, MN), followed by a powder-
a moated petri dish until 10-15 min post-application had free latex glove (Diamond Grip, Microflex Corporation,
elapsed. Using forceps, the I. scapularis nymphs were then Reno, NV). A knee-high stocking (Leggs everyday knee
transferred individually from the moated Petri dish to the highs, Winston-Salem, NC) was then placed over the gloved
lower untreated zone of the filter paper. The locations of the hand and arm. A plastic sleeve of polyvinyl was the final
ticks were recorded at 1, 3, 5, 10, and 15 min after the tenth layer affixed over the stocking covered arm. The plastic
A. americanum or I. scapularis nymph grasped the lower sleeve was sealed around the arm by a Velcro™ strip. About
untreated zone of the filter paper. Ticks were considered half-way between the wrist and elbow a 4 x 8 cm opening
repelled if they were in the lower untreated zone at 15 min in the sleeve allowed us to assess mosquito landing and
or if they fell from the filter paper without having crossed biting behavior. Attractive odors emanated from the skin
the upper boundary of the treated zone. surface and attracted mosquitoes to the opening. During
testing, this 32 cm2 open area was covered with extract-
Tick experimental design treated muslin cloth. The order in which treated cloths were
Test solutions and ethanol controls were tested in tested was randomized among volunteers and randomized
random order. A control was run each day that the oils and to minimize any variation due to day-to-day effects.
deet were tested. Ticks were tested in replicates of ten ticks A test started when the arm, sleeve, and cloth were
per combination of concentration and oil or deet. Essential inserted into the mosquito cage. If fewer than four bites
oils from C. funebris and J. chinensis were tested against three were received during the 1 min, the dosage of repellent on
replicates of A. americanum at 0.827, 0.413, 0.206, and 0.103 cloth was considered to have “passed.” A treatment in which
mg/cm2filter paper, and J. communis at 0.827, 0.413, and five bites (out of 500 mosquitoes in the cage) occurred in
0.206 mg/cm2 filter paper. A single concentration (0.103 mg 1 min was considered a failure. Normally, an intermediate
oil/cm2 filter paper) of each of C. funebris, J. communis, and dosage (e.g., 0.187 mg/cm2) was tested first. Depending on
J. chinensis essential oil was tested against three replicates whether this concentration passed or failed, higher or lower
(ten ticks per replicate) of I. scapularis nymphs. treatment concentrations were evaluated with all subjects
To determine the duration of the repellency of C. until each had pinpointed their individual concentration
funebris and J. chinensis, we tested their essential oils (0.827 that produced the 1% (five bite) failure point. If the 1.500 mg/
mg oil/cm2 filter paper) against A. americanum nymphs at 2, cm2 (or highest concentration) on cloth was not efficacious
4, and 6 h after application. Deet, at 0.827 mg oil/cm2 filter (>five bites in 1 min), then the minimum effective dosage
paper, was included in the duration tests for comparison. (MED) was noted as ineffective at the highest concentration
Four replicates (ten ticks per replicate) were tested at 2 tested. Because the mosquitoes fatigue upon repeated
and 4 h post-application and three replicates (ten ticks per exposure to repellent and attractant odors from the arm, a
replicate) at 6 h. limit of ten successive tests were conducted, after which the
caged mosquitoes were allowed a 15 min recovery period.
Vol. 36, no. 2 Journal of Vector Ecology 261

Three male volunteers (two tested twice) and a female methodology was used to model the mosquito larvicidal
volunteer participated in the studies of MED of the oils assay. Permethrin was compared to the essential oils in
(USDA 1977). During a test, one or both volunteers wore the adult mosquito topical assay data (also in a generalized
a patch and tested each patch for 1 min intervals. Patches linear models framework).
were rotated between the volunteers, thus, no patch was
evaluated beyond 10 min after the 3-min drying period to RESULTS
avoid any bias that may result from evaporative loss of the
treatment from the cloth during the duration of the test. The Essential oil composition
subjects provided written informed consent. The protocol Essential oils from leaves of J. communis and wood oils
was approved by the University of Florida Human Use from J. chinensis and C. funebris were characterized and
Institutional Review Board-01 (Study # 636-2005). identified by gas chromatography and gas chromatography/
mass spectrometry. The relative percentages of the
Mosquito larvicidal assay constituents are listed in Table 1. Forty-seven (95.5% total
Larval bioassays were performed as described in oil), 46 (72.6%), and 55 (66.8%) compounds were identified
Pridgeon et al. (2009). Briefly, five Ae. aegypti 1st instar from J. communis, J. chinensis, and C. funebris essential oils,
larvae were placed into individual wells of a 24-well respectively. The major components of each species were:
plate containing 950 µl deionized water and 40 µl of a 2:1 α-pinene (26.9%), α-terpineol (14.0%), linalool (10.9%),
suspension of alfalfa:pot belly pig chow. Chemicals to be limonene (6.2%), and β-pinene (5.2%) for J. communis;
tested were resuspended in DMSO (Sigma Cat. D8418), cuparene (11.3%), δ-cadinene (7.8%), and α-cedrene (4.8%)
for a stock concentration of 50 µg/µl. Six concentrations in for J. chinensis; α-cedrene (16.9%), cedrol (7.6%), β-cedrene
a two-fold serial dilution series in DMSO were tested for (5.7%), and cuparene (5.4%) for C. funebris.
mortality by adding 10 µl of each concentration into the
wells. After 24 h, the number of dead larvae was recorded. Tick repellency
Serial dilutions were continued until 0% mortality was All three essential oils were repellent to A. americanum
observed for each chemical. All concentrations were tested and I. scapularis nymphs but were effective against I.
in triplicate. Controls included negative (untreated), carrier scapularis nymphs at a much lower concentration than
(DMSO), and positive (permethrin). those needed to repel similar percentages of A. americanum.
Figure 1 depicts dose-related responses of A. americanum
Mosquito adult topical assay nymphs to the three oils and to deet. The EC95 values of C.
Stock chemicals prepared from above were diluted funebris, J. chinensis, and J. communis are 0.43, 0.92, and
into acetone for a final concentration of 6.25 µg/µl. Ten 0.51 mg oil/cm2 filter paper, respectively, whereas the EC95
adult Ae. aegypti female mosquitoes, three to five days post- for deet is 0.68 mg oil/cm2 filter paper (Table 2). Among
eclosion, were cold anaesthetized and placed on BioQuip the oils, the EC50 of J. communis (0.288 mg oil/cm2 filter
chill table (Rancho Dominguez, CA) set at 4° C. Using a paper) was the highest, but that of deet was 0.394 (Figure
#1702 Gastight Hamilton syringe mounted onto a Hamilton 1). Cupressus funebris oil, at 0.103 mg oil/cm2 filter paper,
PB600 repeating dispenser (Reno, NV), 0.5 µl of the test repelled all I. scapularis nymphs, and the same concentration
chemical was applied to the dorsal thorax of each insect, of J. chinensis and J. communis oils repelled 90% and 73.3%
with a final dose of 3.125 µg per insect. After treatment, respectively.
mosquitoes were placed in 3.5 oz (0.10 l) plastic cups, The oils of C. funebris and J. chinensis, at 0.827 mg oil/
supplied with 10% sucrose solution, and maintained at 28° cm2 filter paper, repelled ≥80% A. americanum 4 h after
C and 80% relative humidity. All assays were performed in application (Table 3). Although repellent activity for both
triplicate. Controls included negative (untreated), carrier oils remained at 6 h post-application, it clearly showed
(DMSO-acetone), and positive (permethrin). diminished effectiveness.

Statistical analysis Mosquito repellency

The proportion of ticks in the vertical filter paper Neither the oil of C. funebris nor that of J. chinensis
bioassay that were repelled was modeled in the generalized consistently repelled female Ae. aegypti mosquitoes from
linear models framework (as binomial proportions) using biting through cloth treated at the highest dosage tested
the R statistical software glm function, with estimated EC50 (1.500 mg/cm2). However, the oil of J. communis had a MED
and EC95 values computed using the dose.p function in the of 0.057 ± 0.013 mg/cm2 (Table 4).
MASS package. A straight line relationship between the
proportion repelled on the logit scale and concentration was Mosquito toxicity
obtained after a power transformation on concentration for Mortalities of 100, 80, and 67% at 250 ppm and 80, 27,
some essential oils. When results from high concentrations and 40% at 125 ppm in Ae. aegypti larvae were observed for
were dropped, sometimes the proportion repelled was J. chinensis, C. funebris, and J. communis oils, respectively.
slightly lower in the highest concentration than at a previous The concentrations required to produce these levels of
lower concentration. With relatively few concentrations mortality by these oils are significantly higher than the
used, this artificially affects the modeling. The same 0.25 ppb of the commonly used insecticide, permethrin
262 Journal of Vector Ecology December 2011

(synthetic pyrethroid), which produced 67% (SEM 5.2%) cultivated at Waco, TX, and collected from Japan were
mortality. The LD50s and LD95s of the oils and deet for Ae. found by Adams and Li (2008) to be cis-thujopsene (28.4,
aegypti larvae are presented in Table 5. Mortality of adult 8.4%), cedrol (13.7, 39.4%), widrol (9.2, 2.0%), α-cedrene
Ae. aegypti was also negligible, 3.3% at a dose of 3.125 ug (3.6, 3.1%), and β-cedrene (3.5, 0.6%), respectively. These
oil/insect, for all three oils. differences could be due to distillation variation or to
ecological or climatic variables associated with the localities
DISCUSSION where the plants grew.
Cupressus funebris wood oil is characterized by
α-Pinene predominates in most of the leaf oils of the occurrence of four components: cedrol, α-cedrene,
J. communis from countries of the eastern hemisphere β-cedrene, and thujopsene. In our study, α-cedrene (16.9%),
(Butkiene et al. 2006). In contrast, major components of the cedrol (7.6%), and β-cedrene (5.7%) were the major
oil of J. chinensis that we analyzed were cuparene (11.3%), constituents of C. funebris oil. The chemical composition
δ-cadinene (7.8%), α-cedrene (4.8%), and cedrol (3.2%). of our samples resembled those of samples originating in
The main components of wood essential oils of J. chinensis China or Vietnam (Adams and Li 2008, Duquesnoy et al.

deet J. chinensis
1.0

1.0
● ●
●

● ●
0.8

● 0.8
proportion repelled

proportion repelled

● ●
0.6

0.6

● ● ●
0.4

0.4

● ●
0.2

0.2

● ● ●
●
● ●
● ●
0.0

0.0

●
● ●
●
● ● ●

0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0

concentration (mg/cm2) concentration (mg/cm2)

C. funebris J. communis
1.0

1.0

●
●
●
0.8

0.8

●
● ● ●
●
● ● ●
proportion repelled

proportion repelled

●
0.6

0.6

●
●
●
0.4

0.4
0.2

0.2

● ●
● ● ●
● ●
● ●
0.0

0.0

●
● ●
●
●
● ●
●

0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0

concentration (mg/cm2) concentration (mg/cm2)

Figure 1. Responses of A. americanum nymphs to the essential oils of C. funebris, J. chinensis, and J. communis, deet, and an
ethanol control (concentration of zero) in vertical filter paper bioassays. EC50 and EC95 indicated by arrows.
Vol. 36, no. 2 Journal of Vector Ecology 263

Table 1. Composition of essential oils of Juniperus communis, J. chinensis and Cupressus funebris.

RRI Compound Jc % Jch % Cf %

1014 Tricyclene 0.1
1032 α-Pinene 27.0 0.1
1035 α-Thujene 0.2
1076 Camphene 1.2
1118 β-Pinene 5.2 Tr
1132 Sabinene 1.2
1174 Myrcene 2.5
1203 Limonene 6.2 0.1
1213 1,8-Cineole 0.7
1280 p-Cymene 0.8
1384 α-Pinene oxide 0.8
1406 α-Fenchone 0.1
1429 Perillen 0.3
1450 trans-Linalool oxide (Furanoid) 0.3
1466 α-Cubebene 0.1
1478 cis-Linalool oxide (Furanoid) 0.4
1493 α-Ylangene 0.1
1497 α-Copaene 0.4 0.4
1500 Pentadecane Tr
1513 Longicyclene 0.2
1519 1,7-Diepi-α-Cedrene (=α-Funebrene) 0.1 0.4
1532 Camphor 2.2
1538 7,8-Dihydrolinalool 0.1
1541 Modhephene 0.1
1553 Linalool 10.9
1562 Isopinocamphone 0.1
1568 trans-α-Bergamotene 0.1
1577 α-Cedrene 4.8 16.9
1583 Terpinen-1-ol 0.5
1583 Longifolene (=Junipene) 2.5
1591 Bornyl acetate 2.1
1594 1,7-Diepi-β-cedrene (=β-Funebrene) 0.3 1.4
1600 β-Elemene 1.2 0.5
1611 Terpinen-4-ol 1.6
1612 β-Caryophyllene 2.0 1.0
1613 β-Cedrene 1.1 5.7
1641 cis-β-Terpineol 1.9
1644 Widdrene (=Thujopsene) 2.2
1648 Myrtenal 0.5
1661 Alloaromadendrene 0.4
1670 trans-Pinocarveol 0.6
1677 epi-Zonarene 1.1
1682 δ-terpineol 0.5
1687 α- humulene 0.6 0.3 0.2
1688 Selina-4,11-diene (=4,11-Eudesmadiene) 0.4 0.9
1690 α-Acoradiene 0.2 0.4
1690 trans-β-terpineol 0.9
1693 β-Acoradiene 0.3 1.2
1704 γ-Muurolene 0.3 0.8 1.4
1706 α-Terpineol 14.0
1715 γ-Terpineol 1.4
1718 β-Alaskene 0.4 1.0
1719 Borneol 0.6
Continued on next page
264 Journal of Vector Ecology December 2011

Table 1. Continued.
RR1 Compound Jc % Jch % Cf %
1725 Verbenone 0.3
1725 β-=Chamigrene 0.9
1729 Zonarene 0.4 0.5
1740 α-Muurolene 0.4 1.6 2.0
1742 β-Selinene 0.8 1.5
1744 α-Selinene 1.0 1.7
1747 α-Alaskene 0.7 3.3
1751 Carvone 0.2
1759 α-Cuprenene 0.3 1.1
1762 α-Chamigrene 0.4 0.6
1771 γ-Bisabolene 1.2
1773 δ-Cadinene 7.8 2.7
1776 γ-Cadinene 0.3 0.4 0.9
1786 ar-Curcumene 1.0 2.5
1801 β-Cuprenene 0.8 2.0
1804 Myrtenol 0.4
1807 α-Cadinene 0.3 0.5
1845 trans-Carveol 0.2
1849 Cuparene 11.3 5.4
1849 Calamenene 4.3 1.0
1864 p-Cymen-8-ol 0.4
1918 β-Calacorene 0.7 0.1
1941 α-Calacorene 2.7 1.5
1949 (Z)-3-Hexenyl nonanoate 0.5
2001 Isocaryophyllene oxide 0.8
2008 Caryophyllene oxide 2.6
2050 (E)-Nerolidol 0.1
2051 Gleenol 1.2 0.2
2071 Humulene epoxide-II 0.5
2080 Cubenol 1.3 0.3
2080 Junenol (=Eudesm-4(15)-en-6-ol) 0.1
2088 1-epi-Cubenol 2.8 0.3
2143 Cedrol 3.2 7.6
2178 Widdrol 0.8
2185 γ-Eudesmol 0.8
2200 Pimara-8,15-diene 0.6
2209 T-Muurolol 0.9 0.3
2217 α-Cedrenal 0.1 0.2
2219 δ-Cadinol (=alpha-muurolol) 0.6
2232 α-Bisabolol 1.3 0.5
2250 α-Eudesmol 0.1
2255 α-Cadinol 0.1
2256 Cadalene 1.8 0.4
2357 14-Hydroxy-β-caryophyllene 0.6
2373 14-Oxo-α-muurolene 0.5
2478 14-Hydroxy-α-humulene 0.4
2501 8,13-Abietadiene 0.1
2524 Abietatriene 0.2
2568 14-Hydroxy-α-muurolene 0.3
2607 14-Hydroxy-δ-cadinene 0.2
2694 14-Hydroxy-calamenene 0.1
Total 95.5 66.8 72.6

Jc: J communis; Jch: J. chinensis; Cf: C. funebris; RRI: Relative retention indices calculated against
n-alkanes; % calculated from FID data; Tr: trace (< 0.1 %).
Vol. 36, no. 2 Journal of Vector Ecology 265

Table 2. Concentrations of essential oils of J. chinensis, J. communis, C. funebris, and deet estimated to repel 50 and
95% of A. americanum nymphs in vertical filter paper bioassays.

Concentrationa
Repellent 95% CI Intercept (±SE) Slope (±SE)
(Power Transformation)
J. communis EC500.288 (none) 0.243 – 0.334 -3.855 (±0.375) 13.362 (±1.544)
EC95 0.509 (none) 0.424 - 0.594
J. chinensis EC50 0.268 (sq rt) 0.206 – 0.331 -3.477 (±0.305) 6.706 (±0.662)
EC95 0.917 (sq rt) 0.788 – 1.046
C. funebris EC50 0.209 (none) 0.165 – 0.253 -3. 582 (±0.323) 11.589 (±1.258)
EC95 0.465 (none) 0.424 – 0.594
Deet EC50 0.394 (none) 0.333 – 0.455 -4.011 (±0.403) 10.179 (±1.241)
EC95 0.683 (none) 0.567 – 0.800

a
Concentrations as mg oil or deet/cm2 filter paper.

Table 3. Percent of A. americanum nymphs repelled by C. funebris and J. chinensis

essential oils, and deet at 0.827 mg/cm2 filter paper and ethanol control 2, 4, and 6 h after
test solutions were applied to filter paper.

2 ha 4 ha 6 hb
% (mean ±SD)c % (mean ± SD) % (mean ± SD)
C. funebris oil 90.00 (9.00±1.41) 82.50 (8.25±1.26) 67.67 (6.67±2.31)
J. chinensis oil 95.00 (9.50±1.00) 80.00 (8.00±3.37) 46.67 (4.67±1.16)
Deet 97.50 (9.75±0.50) 97.50 (9.75±0.50) 90.00 (9.00±1.00)
Ethanol 5.00 (0.50±0.58) 0 (0±0) 3.33 (0.33±0.58)
a
Four replicates tested. bThree replicates tested. cMean per replicate of ten ticks.

Table 4. Minimum effective doses (MED)a of J. chinensis, J. communis, and C. funebris essential oils
tested on human volunteers against Ae. aegypti mosquitoes.

Volunteer no. J. chinensis J. communis C. funebris

1 >1.5b 0.011 >1.5
1 >1.5 0.047 >1.5
2 >1.5 0.047 >1.5
2 0.094 0.047 >1.5
3 >1.5 0.094 >1.5
4c 0.187 0.094 >1.5
a
MED in mg/cm2.. bIf ≥5 bites in 1 min exposure to 500 mosquitoes then that compound/dose was
considered a failure. cVolunteer 4 was a female, the other three volunteers males (volunteers 1 and 2
tested twice).
266 Journal of Vector Ecology December 2011

Table 5. Doses of essential oils of J. communis, C. funebris, and permethrin estimated to kill 50 and 95% of Ae. aegypti larvae.
There were not enough intermediate concentrations for J. chinensis to calculate the ED50 and ED95. Intercept and slope values
(±SE) are given for a generalized linear model (with proportion, the dependent variable, on the logit scale).
Concentrationa
Toxicant 95% CI Intercept (±SE) Slope (±SE)
(Power Transformation)
J. communis LD50 163.6 ppm (sqrt) 120.2 – 213.7 -4.596 (±0.870) 0.359 (±0.071)
LD95 440.3 ppm (sqrt) 282.6 – 632.9
C. funebris LD50 263.2 ppm (none) 200.5 – 325.8 -3.726 (±0.718) 0.021 (±0.004)
LD95 298.9 ppm (none) 229.8 – 368.0
Permethrin LD50 0.1894 ppb (sqrt) 0.146 – 0.239 -6.279 (±1.461) 14.426 (±3.381)
LD95 0.4088 ppb (sqrt) 0.273 – 0.571
a
Ten larvae tested for each of six concentrations for each oil, and seven concentrations for permethrin.

2006). cm2) was not quite as repellent as technical (85%) deet,

Among the major constituents of the three oils, whose MED typically ranged from 0.005-0.023 mg/cm2 for
α-terpineol (14.0% J. communis) and linalool (10.9% J. volunteers participating in recent studies by Tabanca et al.
communis) are known to repel A. americanum nymphs, (2010). Just over 50% of the composition of the J. communis
as do carveol and carvone, less plentiful J. communis oil is α-pinene (27%), α-terpineol (14%), and linalool
components (Weldon et al. 2011). Terpinen-4-ol (1.6% in (11%). Linalool has been well characterized as a repellent
J. communis) has been found to be somewhat repellent to I. and attraction-inhibitor of mosquitoes (Hwang et al. 1985,
scapularis (Dietrich et al. 2006) and A. americanum nymphs Traboulsi et al. 2005, Park et al. 2005, Kline et al. 2003).
(Weldon et al. 2011). Singh et al. (1984) reported the toxicity of cedarwood oil
The oils of C. funebris and J. communis are comparable to mosquitoes. However, they tested oil from the deodar or
to deet in their effectiveness in repelling A. americanum, Himalayan cedar, Cedrus deodara Roxb., which is classified
but have steeper dose response curves than deet and J. in the Pinaceae, whereas C. funebris, J. communis, and J.
chinensis oil (Figure 1) and lower EC50s and EC95s than deet. chinensis are in the Cupressaceae. Both the Pinaceae and
The only oils, C. funebris and J. chinensis, tested for duration Cupressaceae belong to the order Pinales.
of repellency were effective (≥80% A. americanum repelled) Perhaps most encouraging is that an oil (J. communis)
for 4 h after application. Deet retained 90% repellency at 6 comprising multiple compounds exhibited a MED against
h post-application, at which time C. funebris and J. chinensis a mosquito, Ae. aegypti, and an EC95 against a tick, A.
oils had declined to 67.7 and 46.7% repellency. The oils americanum, that resembled the values obtained for a single
are clearly not ephemeral and formulation chemistry can pure synthetic repellent (deet). This indicates the presence
extend the longevity of repellent activity should they be of at least one good repellent and probably multiple repellent
developed into commercial repellent products. compounds that act synergistically to produce the observed
The tendency of I. scapularis nymphs to be repelled repellency. Panella et al. (1997) tested extracts from six
by considerably lower concentrations of repellent than species of the Cupressaceae for toxicity to I. scapularis
A. americanum nymphs has been reported for several larvae and nymphs, and found that the extract from Alaska
chemicals (Carroll et al. 2004, 2005, 2007), and was again yellow cedar, Chamaecyparis nootkatensis (D. Don) Spach,
observed in our bioassays with the three oils. was the most effective extract against larvae, and the extract
Because C. funebris and J. chinensis oils were not from eastern red cedar, Juniperus virginiana L., most
efficacious as repellents or toxicants against Ae. aegypti effective against nymphs. Among the compounds identified
at the highest concentrations, neither of these oils nor from Alaska yellow cedar essential oil, the sesquiterpene
their major constituents merit additional examination nootkatone and monoterpene carvacrol were found notably
as repellents or insecticides for Ae. aegypti. The oil of C. toxic to I. scapularis nymphs (Panella et al. 2005). Dietrich
funebris was one of 38 oils tested for repellency against Ae. et al. (2006) found that nootkatone, and carvacrol were
aegypti (Trongtokit et al. 2005), and found to be ineffective similar to deet in their capacity to repel I. scapularis. In
at 30 min post-application for 10% and 50% dilutions; there field tests, nootkatone effectively suppressed populations of
was only one case where repellency was noted at 30 min I. scapularis and A. americanum (Dolan et al. 2009). The
post-application. Neither of the Juniperus species reported progress made in developing nootkatone and carvacrol as
here was tested by Trongtokit (2005). Amer and Mehlkorn anti-tick compounds (Dolan et al. 2009) is an incentive
(2006) reported that in a study with human volunteers, the for continued research on the constituent compounds of J.
essential oil of J. communis (plant source Austria) repelled communis, and for further efforts in repellent and toxicant
43.2% of Ae. aegypti at 210 min after application. discovery among the essential oils (and constituents) of
In our study, the oil of J. communis (0.057 ± 0.013 mg/ Cupressaceae.
Vol. 36, no. 2 Journal of Vector Ecology 267

Acknowledgments pyrethroids in public health and vector pest control. J.

Pest Sci. 27: 361-374.
This study was supported in part by a grant from CDC. 2002. Lyme disease. Department of Health and
the Deployed War-Fighter Protection (DWFP) Research Human Services, Centers for Disease Control and
Program and the U.S. Department of Defense through the Prevention, Ft. Collins, CO, 12 pp.
Armed Forces Pest Management Board (AFPMB). We thank Childs, J.E. and C.D. Paddock. 2003. The ascendancy of
J. L. McCrary and A. S. Khan, USDA, ARS, Invasive Insect Amblyomma americanum as a vector of pathogens
Biocontrol and Behavior Laboratory, Beltsville Agricultural affecting humans in the United States. Annu. Rev.
Research Center, Beltsville, MD, for carefully performing Entomol. 48: 307-337.
many of the tick bioassays. We are grateful to USDA, Dayan F.E., C.L. Cantrell, and S.O. Duke. 2009. Natural
ARS, Knipling-Bushland U.S. Livestock Insects Research products in crop protection. Bioorgan. Med. Chem. 14:
Laboratory, Kerrville, TX, for providing A. americanum for 4022-4034.
bioassays. Debboun, M., S.P. Frances, and D. Strickman. 2007. Insect
Repellents: Principles, Methods, and Uses, CRC Press,
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